Semicarbazone derivatives as urease inhibitors: Synthesis, biological evaluation, molecular docking studies and in-silico ADME evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.03.029

A series of hydrazinecarboxamide derivatives were synthesized and examined against urease for their inhibitory activity. Among the series, the 1-(3-fluorobenzylidene)semicarbazide (4a) (IC₅₀ = 0.52 ± 0.45 μM), 4u (IC₅₀ = 1.23 ± 0.32 μM) and 4h (IC₅₀ = 2.22 ± 0.32 μM) were found most potent. Furthermore, the molecular docking study was also performed to demonstrate the binding mode of the active hydrazinecarboxamide with the enzyme, urease. In order to estimate drug likeness of compounds, in silico ADME evaluation was carried out. All compounds exhibited favorable ADME profiles with good predicted oral bioavailability.

Full text of DOI:10.1016/j.bioorg.2018.03.029

Accepted Manuscript
Semicarbazone Derivatives as Urease Inhibitors: Synthesis, Biological Evalu-
ation, Molecular Docking Studies and In-Silico ADME Evaluation
Syeda Uroos Qazi, Shafiq Ur Rahman, Asia Naz Awan, Mariya al-Rashida,
Rima D. Alharthy, Asnuzilawati Asari, Abdul Hameed, Jamshed Iqbal
PII:
S0045-2068(17)30800-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.029
YBIOO 2319
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
20 October 2017
20 March 2018
31 March 2018
Please cite this article as: S. Uroos Qazi, S. Ur Rahman, A. Naz Awan, M. al-Rashida, R.D. Alharthy, A. Asari, A.
Hameed, J. Iqbal, Semicarbazone Derivatives as Urease Inhibitors: Synthesis, Biological Evaluation, Molecular
Docking Studies and In-Silico ADME Evaluation, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.03.029
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Semicarbazone Derivatives as Urease Inhibitors: Synthesis, Biological Evaluation,
Molecular Docking Studies and In-Silico ADME Evaluation
Syeda Uroos Qazi,a,c Shafiq Ur Rahman,b Asia Naz Awan,c Mariya al-Rashida,d Rima D.
Alharthy,e Asnuzilawati Asari,f Abdul Hameed,a* and Jamshed Iqbalb*
a H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan.
b
Center for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi.
d Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur
Road, Lahore-54600, Pakistan.
e Department of Chemistry, Science and Arts College, Rabigh Campus, King Abdulaziz,
University, Jeddah, Saudi Arabia.
f School of Fundamental Science, Universiti Malaysia Terengganu, 21030 kuala Terengganu,
Terengganu, Malaysia.
Abstract
A series of hydrazinecarboxamide derivatives were synthesized and examined against urease
for their inhibitory activity. Among the series, the 1-(3-fluorobenzylidene)semicarbazide (4a)
(IC50 = 0.52 0.45 µM), 4u (IC50= 1.23 0.32 µM) and 4h (IC50=2.22 0.32 µM) were
found most potent. Furthermore, the molecular docking study was also performed to
demonstrate the binding mode of the active hydrazinecarboxamide with the enzyme urease.
In order to estimate drug likeness of compounds, in-silico ADME evaluation was carried out.
All compounds exhibited favorable ADME profiles with good predicted oral bioavailability.
Keywords: Semicarbazide, Semicarbazones, Urease inhibition, Molecular docking, ADME
evaluation.
*abdul_hameed8@hotmail.com (A. Hameed)
*drjamshed@ciit.net.pk / jamshediqb@googlemail.com (J. Iqbal).

1.
Introduction
Urease (urea amidohydrolase; E.C.3.5.1.5) is a metallo-enzyme bearing two nickel atoms in
its active site and is responsible for the catalytic hydrolysis of urea to ammonia and
carbamate. The carbamate species is further decomposed to ammonia and carbon dioxide.
Urease is a widely spread enzyme found in various organisms such as plants, fungi, and a
diverse range of prokaryotes [1-5]. The overexpression of pathogenic urease results in the
increased production of ammonia which further leads to elevated pH level causing negative
consequences in human health i.e. ulcer, and gastric infections [6]. The increased pH in
stomach due to urease hyperactivity provides enduring environment for rapid growth of
pathogenic bacteria i.e. Helicobacter pylori (H. pylori) that plays an important role in
inducing gastric and peptic ulcers [7]. Urease also has role in the virulence of many other
infections including infectious stones in kidney, urinary tract infections, hepatic coma etc. [8-
10].
Figure-1: Structure of previous thiosemicarbazones, thiourea and aldehyde/ketone based
semicarbazones.
To modulate urease activity, a successful therapeutic strategy is the application of small
molecules as urease inhibitors to overcome gastric complications due to urease hyperactivity
[7]. Until now, an array of urease inhibitors have been reported, however, few of them
proceed to advanced level of drug development as urease inhibitors. Previously, we have
reported thiosemicarbazones [11] and N-phenyl thiosemicarbazones [12] as urease inhibitors

due to their close resemblance with thiourea which has been used as reference in urease
inhibition assay. In the continuation of our work towards urease inhibitors, herein, we report
the synthesis of a series of semicarbazones 4a-4u, with better urease inhibitory activity in
comparison to thiourea used as reference inhibitor (Figure-1).
Semicarbazones have been widely studied in medicinal chemistry due to their broad array of
biological activities [13]. The semicarbazone based compounds have shown anti-convulsant
[14-17], antimicrobial [18], pesticidal [19], sedative [20], anti-protozoal, anticancer activity
etc. [21]. In the current study, we have synthesized a series of semicarbazones 4a-4u to
evaluate them as urease inhibitors.
Scheme-1: Synthesis of semicarbazones 4a-4u.
Table-1: In vitro urease inhibitory activities of semicarbazones 4a-4u.
Comp.
No.
IC50 SEM (µM)a
Entry
Structures
1
0.52 0.45
4.54 0.15
7.76 0.55
4a
2
3
4b
4c

4
5
9.31 0.19
7.45 0.91
4d
4e
6
7
5.65 0.17
4.79 0.12
4f
4g
8
9
2.22 0.32
11.6 0.91
3.63 0.67
4h
4i
10
4j
11
12
13
14
15
16
6.59 0.13
13.9 0.56
6.45 0.36
5.24 0.84
9.05 0.51
12.2 0.37
4k
4l
4m
4n
4o
4p

17
18
19
20
21
6.52 0.11
8.30 0.62
28.9 0.98
6.21 0.34
4q
4r
4s
4t
1.23 0.38
21.0 0.10
4u
Thioureab
a Half maximal inhibitory concentration.
b Standard inhibitor for urease inhibition.
2.
Results and discussion
Chemistry
2.1
The synthesis of semicarbazones was carried out as shown in Scheme-1. The solution of
semicarbazide hydrochloride with sodium acetate in ethanol was reacted with several
substituted aromatic aldehydes and ketones which gave respective semicarbazones in
excellent yields i.e. 95-99.9% in case of aldehydes, while 80-90% in case of ketones. The
desired compounds were obtained by evaporating the solvent and recrystallization from
suitable solvent i.e. ethanol. Characterization of semicarbazones 4a-4u was done via different
spectroscopic techniques including IR, MS and 1H NMR spectroscopy.
A typical semicarbazone 4b (E)-2-(3-fluorobenzylidene)hydrazine-1-carboxamide has been
selected as an example for the description of structural analysis by different spectroscopic
technique i.e. IR and 1H NMR spectroscopy. In IR spectrum, the presence of stretching bands
around 3471 cm-1, 1698 cm-1, and 1582 cm-1 showed the presence of -NH2, -CH=N, and -
1
CONH2. H NMR spectrum of compound 4b was recorded using DMSO on a Bruker AM
400 MHz instrument. Characteristic singlets at δH 10.32 ppm and 6.55 ppm were observed for
NH and NH2 respectively. The CH of semicarbazones moiety appeared as a singlet at δH 7.81

ppm confirming the formation of semicarbazones. The aromatic protons (ArH) of 2 appear
between δH 7.71 and 7.37 ppm.
1.1
Urease Inhibition Assay (in vitro) and Structure Activity Relationship
All of the synthesized semicarbazones 4a-4u were evaluated in vitro for urease inhibitory
activity. Almost all of the synthesized compounds exhibited potent urease inhibitory activity.
All compounds contained various functional groups substituted at the phenyl ring. Thiourea
was used as reference standard. From the series the compound 4a was the most potent with
IC50 value of 0.52 0.45 µM, while second most potent compound was 4u with IC50 value of
1.23 0.38 µM, and the third most potent compound was 4h with IC50 value of 2.22 0.32
µM. Other potent compounds were 4b-4g, 4i-4r, 4f having with IC50 values ranging from
3.63 0.67 µM to 13.9 0.56 µM. The compound 4s only possessed activity less than the
standard inhibitor with IC50 value of 28.9 0.98 µM.
The activity of the compounds was greatly influenced by the change in substitutions at ortho,
meta and para positions of phenyl ring. Compound 4a which possessed fluorine at meta
position with respect to semicarbazide moiety was the most potent with IC50 value of 0.52
0.45 μM, while changing the position of fluorine from meta to para position in compound 4b
resulted in the decline in activity with IC50 value of 4.54 0.15 mM. However, substituting
fluoro group with bromo at meta position and adding a methoxy group at para position, as in
compound 4u improved activity (IC50 = 1.23 0.38 mM) as compared to compound 4b, but
the activity was slightly less than the most potent compound 4a. Furthermore, the compound
4h (IC50 = 2.22 0.32 mM), possessing nitro group at para position showed increased activity
as compared to compound 4b bearing fluoro group at para position, but changing the position
of nitro group from para to ortho, as in compound 4j resulted in decreased activity (IC50
value of 3.63 0.67 mM), which further decreases on changing the position of nitro group to
meta in compound 4i (IC50 = 11.6 0.91 mM).

In case of bromo containing compounds, bromo substituent at ortho position in aldehyde
derived hydrazinecarboxamide 4q (IC50 = 6.52 0.11 mM), and at para position in ketone
derived hydrazinecarboxamide 4e (IC50 = 7.45
compared to when bromo substituent was at meta position in both ketone derived
hydrazinecarboxamide 4r (IC50 8.30 0.62 mM) and aldehyde derived
0.91 mM) showed better activity as
=
hydrazinecarboxamide 4p (IC50 = 12.2 0.37 mM). However, addition of methoxy group at
para position of compound 4p, as in compound 4u resulted in dramatic increase in activity
(Table-1). Replacing bromo group with chloro group at meta position of aldehyde derived
hydrazinecarboxamide 4c resulted in increased activity with IC50 value of 7.76 0.55 mM.
In case of cyano group bearing hydrazinecarboxamide 4n having cyano group at meta
position, better activity (IC50 = 5.24 0.84 mM) was observed than when this group was at
ortho position as in compound 4d (IC50 = 9.31
0.19 mM). The compound 4j which
possessed nitro group at ortho position showed better urease inhibitory activity (IC50 = 3.63
0.67 mM) than all other substitutions at ortho position. Other substituents showed less activity
(Table-1).

2.3
Docking results
To investigate the binding modes, molecular docking of hydrazinecarboxamides against
urease enzyme was performed. It was observed that all of the compounds bind at the active
site of the enzyme and was making interactions with the active site residues of urease. For
each ligand, 30 different conformations were generated against the protein. All the
compounds were found to reside near Ni ions and binding site residues like Gln635, Gly550,
His593, Ala636, Met637, Arg609, His519, Glu493, Asp494, Ala440 and Arg439. The
entrance site of pocket showed electrostatic and polar interactions with all the ligand
molecules as shown in the Figures-2-4.
The most active compound (4a, Figure 2) was noticed to contribute three conventional
hydrogen bonds between the two amide -NH groups and the oxygen of urea moiety with
Arg609, Glu493 and Asp494. For the second most potent inhibitor (4u, Fig. 4), the nitrogen
atom of the semicarbazone moiety (-N=NH-) was found to make hydrogen bond with
Arg609. Whereas the third most potent inhibitor (4h, Fig. 3) was found to form 4 hydrogen
bonds, one between the nitro group of benzene and Gln635 and the other three between the
urea moiety and Arg609, Asp494 and Glu493. Moreover, π-π interactions were also observed
between the active site residues and the compounds.
There is a significant effect of the phenyl ring substituent on the inhibitory activity as
evidenced from the docking study. The hydrogen bond interaction of nitrogen atoms of
semicarbazone moiety with Arg609, was found to be present in docked conformations of all
three docked inhibitors (4a, 4u, 4h). Similarly the hydrogen bond interaction of nitrogen
atoms of semicarbazone moiety with Asp494 and Glu493 were conserved for compounds 4a
and 4h, for compound 4u containing bromo and methoxy substituents on the phenyl ring,
halogen bonding interaction of bromo substituent with Asp633 and Kcx490 (modified amino
acid residue) was observed. Similarly for compound 4h, the nitro substituent was making
hydrogen bonds with Gln635. Hence different interactions observed between the phenyl ring
substituents and the surrounding amino acid residues explain the differences in inhibitory
activity.

Figure-2: Binding mode of compound 4a in urease active site.
Figure-3: Compound 4u docked in the active site of urease enzyme.

Figure-4: Binding mode of compound 4h in the active site of urease.
2.4
In-Silico ADME Evaluation
For in-silico ADME (Adsorption, Distribution, Metabolism, Excretion and Toxicity)
evaluation, and estimation of drug likeness, physicochemical parameters were computed
using FAF-Drugs3 [22]. The selected calculated parameters are given in Table 2. Estimation
of lipophilicity is determined by logP, which is the log of octanol-water partition coefficient.
The logP value should not be greater than 5. As can be seen from the table, all compounds
had favourable logP values in the range. Similarly logD, which is logP at physiological pH of
7.4, and logSW, which is logarithm of compound’s water solubility (calculated based on
EOSOL method [23] were also calculated. All compounds had favourable logD and logSW
values. The estimation of surface contributions of polar fragments is determined by
Topological Polar Surface Area (tPSA). High value of tPSA (> 140 Å2) is linked with low
blood-brain barrier (BBB) penetration, and poor membrane permeability [24]. All compounds
had favourable tPSA values in the range 67-113 (Table 2). According to Lipinski’s rule of 5,
total number of hydrogen bond donors (HBD) should not be more than 5, and total number of
hydrogen bond acceptors (HBA) should not be greater than 10. This standard was also met,

as all compounds had 3-4 HBD and 3-7 HBA. All compounds were predicted to have good
oral bioavailability, as assessed by Veber rule [25]. According to this, the number of rotatable
bonds should be less than or equal to 10 and tPSA ≤ 140 Å, or sum of hydrogen bond donor
and acceptor (HBD+HBA) should be ≤ 12. After applying all rules, the compounds are given
a status either as rejected, if considerably un-favourable ADME profile is predicted, or, in
case of a favourable profile, their status is either accepted or intermediate depending on
extent of favourability. As can be seen from Table 2, none of the compounds were rejected,
and all compounds were given accepted status.

Table-2: In-Silico ADME Evaluation
Comp.
No.
MW
logP
logD
logSW
tPSA
Rotatable bonds
Rigid
bonds
HBD
HBA
Oral Bioavailability
(VEBER)
Status
(I/A)*
181.17
1.37
0.92
-1.91
67.48
2
10
3
4
Good
A
4a
181.17
197.62
188.19
1.37
1.90
1.21
0.92
1.38
0.63
-1.91
-2.34
-1.84
67.48
67.48
91.27
2
2
2
10
10
11
3
3
3
4
4
5
Good
Good
Good
A
A
A
4b
4c
4d
256.10
237.26
1.51
0.76
1.39
0.31
-2.43
-1.66
67.48
85.94
2
4
10
10
3
3
4
6
Good
Good
A
A
4e
4f
179.18
1.14
0.45
-1.74
87.71
2
10
4
5
Good
A
4g
3
3
7
7
Good
Good
A
A
4h
4i
208.17
208.17
0.78
-0.08
0.72
0.72
-1.62
-1.08
113.30
113.30
3
3
12
12
208.17
0.44
0.72
-1.40
113.30
3
12
3
7
Good
A
4j
193.20
193.20
0.20
1.24
0.62
0.62
-1.14
-1.80
76.71
76.71
3
3
10
10
Good
Good
A
A
4k
4l
3
3
3
3
3
3
3
3
4
4
3
5
5
4
5
4
4
4
4
6
6
5
177.20
188.19
209.27
1.49
0.99
1.78
1.29
0.63
1.40
-1.93
-1.70
-2.24
67.48
91.27
92.78
2
2
3
10
11
10
Good
Good
Good
A
A
A
4m
4n
4o
242.07
242.07
256.10
209.20
1.96
2.18
1.51
0.43
1.54
1.54
1.39
0.31
-2.66
-2.79
-2.43
-1.38
67.48
67.48
67.48
96.94
2
2
2
3
10
10
10
10
Good
Good
Good
Good
A
A
A
A
4p
4q
4r
4s
207.19
272.10
0.13
1.26
-2.95
1.39
-1.19
-2.30
107.61
76.71
3
3
11
10
Good
Good
A
A
4t
4u

4.
Conclusion
A series of semicarbazones derivatives (4a-4u) were synthesized and evaluated for inhibitory
activity (in vitro) against enzyme urease. Almost all compounds exhibited potent inhibitory
activity due to the structural similarity of core moiety (semicarbazide) with urease substrate.
Among the series of semicarbazones, 4a exhibited significant urease inhibitory activity.
Furthermore, molecular docking of all compounds was performed against Jack bean urease to
further explore the binding modes of the compounds.
5.
Experimental Work
5.1
Materials and methods
All the starting materials include semicarbazide hydrochloride (Sigma-Alrich ≥99%),
substituted benzaldehydes, substituted acetophenones (≥99%), glacial acetic acid (Sigma-
Alrich ≥99.85%) were purchased from local supplies and used without purification unless
otherwise stated. Distilled water was used for reaction workup purpose as solvent. The
reaction for the synthesis of semicarbazoness was carried out in round bottom flasks and
routine glassware was used for all isolation, purification purposes. Thin layer
chromatography (TLC) was used to monitor reaction by means of silica gel 60 aluminium-
backed plates 0.063-0.200 mm as the stationary phase through ethyl acetate (EtOAc), n-
hexane etc. Chromatograms were envisioned with UV light (254 nm). Infrared spectra IR
(KBr discs) were recorded in the range 4000- 500 cm-1 via Bruker Vector-22 spectrometer.
1H NMR spectra were recorded via Bruker spectrometer 400 MHz as dilute solutions in
suitable deuterated solvent at 25 ºC. The chemical shifts were recorded on the δ-scale (ppm)
1
1
using residual solvents as an internal standard (DMSO; H 2.50, and CHCl3; H 7.26,).
Coupling constant were calculated in Hertz (Hz) and multiplicities were labelled as s
(singlet), d (doublet), t (triplet), q (quartet), quint (quintet) and the prefixes br (broad) or app
(apparent) were used. Mass spectra (EI+) were recorded at Finnigan MAT-321A, Germany.
Melting points of synthesized compounds were determined by means of a Stuart™ melting
point SMP3 apparatus.

5.2
Chemistry
5.2.1 General procedure for the synthesis of semicarbazones (1-21)
A round bottom oven dried flask was charged with aqueous solution of sodium acetate and
semicarbazide hydrochloride. Sequentially, the solution of different substituted aldehydes or
acetophenones in ethanol was added dropwise to the flask at room temperature. The resulting
mixture was stirred until the complete consumption of starting materials. Precipitates of
semicarbazones were formed within 5-10 min in case of aldehydes, while in 10-30 min in
case of ketones. Progress of reaction was monitored through TLC analysis (Scheme 1). The
obtained material was purified via recrystallization from ethanol.
5.2.2 Characterization data of semicarbazones
o
1-(3-Fluorobenzylidene)semicarbazide (4a) [26] Yield: 96%, M.P. 227-229 C;IR (KBr,
cm-1): 3471 (NH), 1698 (C=O), 1586 (CONH2) , 1122, 780; EI-MS: m/z (rel. abund. %), 181
(M+, 25.7), 137 (100), 121 (52.1), 111 (45.6); 1H NMR (400 MHz, DMSO-d6): δH 10.32 (1H,
brs, NH), 7.81 (1H, s, CH), 7.70 (1H, app dq, J = 10.4, 1.2 HZ, ArH), 7.44 (1H, t, J = 7.6 Hz,
ArH), 7.41-7.37 (1H. m, ArH), 7.17-7.11 (1H, m, ArH), 6.56 (2H, s, NH2).
o
1-(4-Fluorobenzylidene)semicarbazide (4b) Yield: 99%, M.P. 228-232.3 C (230.5-231.5
°C) [26]; IR (KBr, cm-1): 3463 (NH2), 1670 (C=O), 1601 (CONH2), 1238, 833; 1H NMR (400
MHz, DMSO-d6): δH 10.24 (1H, brs, NH), 7.83 (1H, s, CH), 7.78 (2H, dd, J = 8.8, 5.6 Hz,
ArH), 7.22 (2H, app t, J = 9.2 Hz, ArH), 6.50 (2H, brs, NH2).
1-(3-Chlorobenzylidene)semicarbazide (4c)] Yield: 99.2%, M.P. 230-234 oC (230 °C) [27];
IR (KBr, cm-1): 3467 (NH2), 1702 (C=O), 1591 (CONH2), 1139. 1HNMR (400 MHz, DMSO-
d6): δH 10.35 (1H, brs, NH), 7.93 (1H, app s, ArH), 7.81 (1H, s, CH), 7.61 (1H, dt, J = 6.4,
2.4 Hz, ArH), 7.42-7.37 (2H, m, ArH), 6.60 (2H, brs, NH2).
o
1-(2-Cyanobenzylidene)semicarbazide (4d) Yield: 95%, M.P. 237-238 C; IR (KBr, cm-1):
3439 (NH2), 2222 (C≡N), 1707 (C=O), 1595 (CONH2), 765. 1H NMR (400 MHz, DMSO-d6):
δH 10.68 (1H, brs, NH), 8.14 (1H, d, J = 8.0 Hz, ArH), 8.11 (1H, s, CH), 7.86 (1H, dd, J =
7.6, 0.8 Hz, ArH), 7.86 (1H, td, J = 7.6, 1.2 Hz, ArH), 7.86 (1H, td, J = 7.6, 1.2 Hz, ArH),
6.62 (2H, brs, NH2).
1-(1-(4-Bromophenyl)ethylidene)semicarbazide (4e) Yield: 86%, M.P. 213-215o C (202-
203 °C) [26]; IR (KBr, cm-1): 3480 (NH2), 1720 (C=O), 1569 (CONH2), 827; EI-MS: m/z

1
(rel. abund. %): 255.1 (M+, 39.7), 240.1 (7.9), 213 (100), 197 (51.6); H NMR (400 MHz,
DMSO-d6): δH 9.36 (1H, brs, NH), 7.79 (2H, d, J = 8.8 Hz, ArH), 7.52 (2H, d, J = 8.8 Hz,
ArH), 6.50 (2H, brs, NH2), 2.15 (3H, s, CH3).
o
1-(1-(2,4-Dimethoxyphenyl)ethylidene)semicarbazide (4f) Yield: 90%, M.P. 204-205 C;
IR (KBr, cm-1): 3430 (NH2), 1689 (C=O), 1586 (CONH2); EI-MS: m/z (rel. abund. %): 237.2
1
(M+, 40.7), 193.1 (23.3), 178.1 (8.7), 163.1 (100); H NMR (400 MHz, DMSO-d6):δH 9.08
(1H, s, NH), 7.26 (1H, d, J = 8.4 Hz, ArH), 6.56 (1H, d, J = 2.0 Hz, ArH), 6.50 (1H, dd, J =
8.4, 2.4 Hz, ArH), 6.22 (2H, brs, NH2), 3.77 (3H, s, OCH3), 3.76 (3H, s, OCH3), 2.05 (3H, s,
CH3).
1-(2-Hydroxybenzylidene)semicarbazide (4g) Yield: 97% , M.P. 236-238 oC (231 °C) [28];
IR (KBr, cm-1): 3468 (NH2), 1677 (C=O), 1597 (CONH2), 1442; EI-MS: m/z (rel. abund. %):
1
179.1 (M+, 100), 162.1 (26.6), 135.1 (54.8), 119 (92.6), 107(43.4); H NMR (400 MHz,
DMSO-d6): δH 10.15 (1H, s, ArOH), 9.95 (1H, brs, NH), 8.13 (1H, s, CH=N), 7.73 (1H, dd, J
= 7.6, 1.2 Hz, ArH), 7.15 (1H, td, J = 7.2, 1.6 Hz, ArH), 6.82 (1H, app dd, J = 7.6, 0.4 Hz,
ArH), 6.80 (1H, t, J = 7.6 Hz, ArH), 6.37 (2H, brs, NH2).
o
1-(4-Nitrobenzylidene)semicarbazide (4h) Yield: 90%; M.P. 215-216 C (220-221 °C)
[29]; IR (KBr, cm-1): 3449(NH2), 1681.7(NO2), 1618(C=O), 1528(CONH2), 1347.2. 1H NMR
(400 MHz, DMSO-d6): δH 10.62 (1H, brs, NH), 8.21 (2H, d, J = 8.8 Hz, ArH), 8.09 (2H, d, J
= 9.2 Hz, ArH), 7.94 (1H, s, CH=N), 6.69 (2H, brs, NH2).
o
o
(E)-1-(3-Nitrobenzylidene)semicarbazide (4i)Yield: 94%, M.P. 240-242 C (238-240 C)
[27]; IR (KBr, cm-1): 3465 (NH2), 1715 (C=O), 1584 (CONH2), 1532, 1349; EI-MS: m/z (rel.
abund. %): 208.2 (M+, 10.5), 164.2 (27.8), 147 (20.2), 118.1 (100), 89 (77.6); 1H NMR (400
MHz, DMSO-d6): δH 10.45 (1H, s, NH), 8.54 (1H, s, CH=N), 8.16 (1H, app td, J = 8.0, 2.0
Hz, ArH), 7.93 (1H, s, ArH), 7.65 (1H, t, J = 8.0 Hz, ArH), 6.64 (2H, brs, NH2).
1-(2-Nitrobenzylidene)semicarbazide (4j) Yield: 95% , M.P. 254-256oC (250-252 °C) [27];
1
IR (KBr, cm-1): 3464 (NH2), 1734 (C=O), 1597 (CONH2), 1524, 135. H NMR (400 MHz,
DMSO-d6): δH 10.62 (1H, brs, NH), 8.34 (1H, dd, J = 7.6, 1.2 Hz, ArH), 8.23 (1H, s,
CH=N), 7.80 (1H, dd, J = 8.0, 1.2 Hz, ArH), 7.72 (1H, app t, J = 7.6 Hz, ArH), 7.58 (1H, app
td, J = 8.0, 1.2 Hz, ArH), 6.62 (2H, brs, NH2).

1-(4-Methoxybenzylidene)semicarbazide (4k) Yield: 100%, M.P. 211-214 oC (213-215 °C)
[29]; IR (KBr, cm-1): 3454.8(NH2), 1688.2(C=O), 1603 (CONH2), 1252; EI-MS: m/z (rel.
abund. %): 193.1(M+,37.4), 149.1(36.4), 133(100), 120(4.9), 108(19.5); 1H NMR (400 MHz,
DMSO-d6): δH 10.05 (1H, NH), 7.77 (1H, s, CH=N), 7.62 (2H, d, J = 8.8 Hz, ArH), 6.92
(2H, d, J = 8.8 Hz, ArH), 6.38 (2H, brs, NH2).
1-(3-Methoxybenzylidene)semicarbazide (4l) Yield: 100%, M.P. 220-222 oC (224-226 oC)
1
[27]; IR (KBr, cm-1): 3461.7 (NH2), 1688 (C=O),1601 (CONH2), 1268. H NMR (400 MHz,
DMSO-d6): δH 10.25 (1H, brs, NH), 7.80 (1H, s, CH=N), 7.34 (1H, app t, J = 2.0, Hz, ArH),
7.29 (1H, app t, J = 7.6 Hz, ArH), 7.21 (1H, app d, J = 7.6 Hz, ArH), 6.93-6.90 (1H, m,
ArH), 6.53 (2H, brs, NH2), 3.80 (3H, s, OCH3).
2-(2-Methylbenzylidene)hydrazine carboxamide (4m) [26] Yield: 98%, M.P. 219.3-220.2
oC, IR (KBr, cm-1): 3469 (NH2), 1698 (C=O), 1582.8 (CONH2), 747; EI-MS: m/z (rel. abund.
1
%): 177(M+, 28.9), 161.1 (1.9), 117 (100), 106 (16.8), 90 (29.1); H NMR (400 MHz,
DMSO-d6): δH 10.15 (1H,s,NH), 8.13 (1H,s,CH=N), 7.90 (1H, app d, J = 7.6, 2.0 Hz, ArH),
7.22-7.16 (3H, m, ArH), 6.40 (2H, s, NH2), 2.35 (3H, s, CH3).
1-(3-Cyanobenzylidene)semicarbazide (4n) Yield: 90%, M.P. 241-242 oC; IR (KBr, cm-1):
3457 (NH2), 2230 (C≡N), 1699 (C=O), 1593 (CONH2); EI-MS: m/z (rel. abund. %): 256.1
(M+,44.3), 224.1 (5), 192 (27.7), 160 (30.8), 128 (31.1), 111.9 (5.2), 95.9 (17.5), 63.9 (100);
1H NMR (400 MHz, DMSO-d6): δH 10.41 (1H,S,NH), 8.35 (1H, s, CH=N), 7.96 (1H, d, J=
8.0 Hz, ArH), 7.82 (1H, s, ArH), 7.75 (1H, d, J = 7.6 Hz, ArH),7.56 (1H, t, J = 7.6 Hz, ArH),
6.64 (2H, s, NH2).
1-(4-(Methylthio)benzylidene)semicarbazide (4o)Yield: 100 %, M.P. 221-223 oC (218-219
oC) [30], IR (KBr, cm-1): 3458 (NH2), 1686 (C=O), 1601 (CONH2); EI-MS: m/z (rel. abund.
1
%): 209.2 (M+, 38.9), 192 (1.5), 165.1 (21.2), 149.1 (100), 136.1 (9.2); H NMR (400 MHz,
DMSO-d6): δH 10.16 (1H, s, NH), 7.77 (1H, s, CH=N), 7.63 (2H, d, J = 8.4 Hz, ArH), 7.23
(2H, d, J = 8.8 Hz, ArH),, 6.43 (2H, s, NH2), 2.48 (3H, s SCH3).
o
1-(3-Bromobenzylidene)semicarbazide (4p) [26,31] Yield: 99%, M.P. 234-235 C; IR
(KBr, cm-1): 3465 (NH2), 1702 (C=O), 1592 (CONH2), 647. 1H NMR (400 MHz, DMSO-d6):
δH 10.36 (1H, brs, NH), 8.07 (1H, app t, J = 1.6 Hz, ArH), 7.80 (1H, s, CH=N), 7.64 (1H, d, J
= 8.0 Hz, ArH), 7.53-7.50 (1H, m, ArH), 7.34 (1H, t, J = 7.8 Hz, ArH), 6.61 (2H, brs, NH2).

1-(2-Bromobenzylidene)semicarbazide (4q) [26] Yield: 100%, M.P. 232-233 oC; IR (KBr,
1
cm-1): 3470 (NH2), 1727 (C=O), 1597 (CONH2), 743, 624. H NMR (400 MHz, DMSO-d6):
δH 10.52 (1H, brs, NH), 8.20 (1H, s, CH=N), 8.81 (1H, dd, J = 8.0, 2.0 Hz, ArH), 7.63 (1H,
dd, J = 8.0, 1.2 Hz, ArH), 7.39 (1H, app t, J = 7.0 Hz, ArH), 7.28 (1H app td, J = 8.0, 2.0 Hz,
ArH), 6.58 (2H, brs, NH2).
o
1-(1-(3-Bromophenyl)ethylidene)semicarbazide (4r) Yield: 99%, M.P. 238.8-241.2 C
1
(229.3-231.3 °C [32]); IR (KBr, cm-1): 3464 (NH2), 1709 (C=O), 1582 (CONH2), 614. H
NMR (400 MHz, DMSO-d6): δH 9.41 (1H, brs, NH), 8.07 (1H, t, J = 2.0 Hz, ArH), 7.82 (1H,
app dt, J = 6.4, 0.8 Hz, ArH), 7.54-7.51 (1H, m, ArH), 7.32 (1H, t, J = 7.8 ArH), 6.58 6.58
(2H, brs, NH2), 2.17 (3H, s, CH3).
o
1-(4-Hydroxy-3-methoxybenzylidene)semicarbazide (4s) Yield: 94%, M.P. 214-216 C
(218.3-219.4 °C [33]); IR (KBr, cm-1): 3516 (NH2), 3463 (OH), 1688 (C=O), 1604 (CONH2);
EI-MS: m/z (rel. abund. %): 209.2 (M+,54.9), 192.1 (4.8), 165.2 (46.6),149.1 (100), 134
(22.2); 1H NMR (400 MHz, DMSO-d6): δH 9.99 (1H, s, OH), 9.26 (1H, brs, NH ), 7.70 (1H,
s, CH=N), 7.35 (1H, d, J = 1.8 Hz, ArH), 6.96 (1H, dd, J= 8.0, 1.6 Hz, ArH), 6.74 (1H, d, J
= 8.0 Hz, ArH), 6.41 (2H, s, NH2),3.80 (3H, s, OCH3).
2-((2-Carbamoylhydrazono)methyl)benzoic acid (4t) Yield: 99%, M.P. 232.3-233.3 oC; IR
1
(KBr, cm-1): 3478 (NH2), 3183 (OH), 1707 (C=O), 1601 (CONH2), 1310. H NMR (400
MHz, DMSO-d6): δH 13.21 (1H, brs, ArCO2H), 10.42 (1H, brs, NH), 8.58 (1H, s, CH=N),
8.19 (1H, app dd, J = 7.6, 0.8 Hz, ArH), 7.81 (1H, dd, J = 6.4, 1.2 Hz, ArH), 7.54 (1H, app
td, J = 8.0, 1.2 Hz, ArH), 7.43 (1H, td, J = 8.0, 1.6 Hz, ArH), 6.58 6.58 (2H, brs, NH2).
2-(3-Bromo-4-methoxybenzylidene)hydrazinecarboxamide (4u) Yield: 85%, M.P. 238.5-
241.6 oC; IR (KBr, cm-1): 3463 (NH2), 1699 (C=O), 1596 (CONH2), 1265; EI-MS: m/z (rel.
1
abund. %): 271.1 (M+, 20), 254.1 (2.3), 229.1 (25.5), 213.1 (100), 186 (6.3); H NMR (400
MHz, DMSO-d6): δH 10.14 (1H, s, NH), 8.07 (1H, d, J = 2.0 Hz, ArH), 7.73 (1H, s, CH=N),
7.58 (1H, dd, J = 8.4, 2.0 Hz, ArH), 7.08 (1H, d, J = 8.8 Hz, ArH), 6.49 (2H, s, NH2), 3.86
(3H, s, OCH3).
Acknowledgment
J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW),
The Hague, The Netherlands and Higher Education Commission of Pakistan through Project
No. 20-3733/NRPU/R&D/14/520 for the financial support.

Supplementary material
Supplementary information includes the in vitro urease inhibition assay protocol, docking
protocol, and the NMR spectra of some semicarbazones. Supplementary data associated with
this article can be found, in the online version, at http//
6.
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Graphical abstract

Highlights
ꢀ A series of semicarbazones has been synthesized.
ꢀ Semicarbazones were evaluated as urease inhibitor for potential lead.
ꢀ Molecular docking and In-Silico ADME evaluation.